Screening and Laboratory Diagnosis of Autoimmune Diseases Using Antinuclear Antibody Immunofluorescence Assay and Specific Autoantibody Testing Paul P. Doghramji, MD

Educational support provided by Quest Diagnostics, Inc.

Introduction Autoimmune diseases occur when the immune system

Laboratory screening and diagnostic testing for disease classification

attacks the normal tissue within joints, vasculature,

The recommended ANA screen approach uses HEp-2

and other organ systems, causing inflammation, pain,

human tissue culture cells in an IFA. In this assay, the

diminished mobility, fatigue, and other non-specific

patient’s blood sample is mixed with HEp-2 cells fixed

symptoms.1 The strong overlap of signs and symptoms

to a slide. ANAs present in the sample react with the

among the autoimmune diseases can lead to delays in

cells and treatment with a fluorescent anti-human IgG

diagnosis and appropriate treatment. According to a

antibody allows visualization of antibody binding under

survey by the Autoimmune Diseases Association, it takes

fluorescence microscopy. This test screens for a large

up to 4.6 years and nearly 5 doctor visits to receive a

number of known autoantibodies, approximately 150,

proper autoimmune disease diagnosis.2

directed against nuclear antigens and cell cytoplasm.

Laboratory testing, in addition to clinical assessment, is necessary for differential diagnosis and disease classification

A positive screen result is followed by evaluation of antibody titer and pattern (consult side bar below).

of autoimmune diseases. However, research shows that

With a positive ANA IFA screen and titer, the clinician

primary care physicians tend to overuse common

needs to determine the root cause of the positivity. This

autoantibody tests for autoimmune conditions, which can

can be accomplished through a reflex to an algorithm

limit the positive predictive value and clinical utility of such

of specific antibody tests to help identify autoantibodies

testing.3 To facilitate appropriate referral to specialists,

associated with specific autoimmune diseases.

if necessary, laboratory testing should be reserved for patients who have signs and symptoms consistent with

An ANA reflex algorithm tests for specific antibodies in a

one or more autoimmune disease (Table 1).

clinically logical sequence. With a combination of ANA IFA plus a reflex algorithm of specific antibody testing, positive

The antinuclear antibody (ANA) immunofluorescence

results automatically reflex to a tier of disease-specific

assay (IFA) is a first-line screening test for patients with

autoantibodies. Testing begins with the most prevalent

a suspected autoimmune disease. This test is the gold

autoimmune diseases and continues until a positive result

standard because of its high sensitivity compared to

is found, or all tests in the algorithm have returned a

other assays.4,5 Positive results should prompt clinicians

negative result. This algorithm/reflex approach provides the

to continue investigating the cause of a positive ANA IFA

clinician with a rational approach to confirming a diagnosis

and narrow the field of potential culprits. The following

in a patient with a suspected autoimmune disease, with a

describes how ANA IFA in combination with specific

single blood draw.

autoantibody testing can be used in the differential diagnosis of a suspected autoimmune disease.

Titer and Pattern If the ANA IFA screen is positive, testing for antibody titer and pattern can help evaluate the presence and type of autoantibody disease. ANA titers are determined by diluting the liquid portion of the blood sample in saline at a ratio of 1:40 to 1:1280. The titer is thus the highest dilution that yields a positive ANA result. Any titer above 1:40 is considered positive, and titers above 1:80 are consistent with an autoimmune disease. To assess the nuclear and cytoplasmic staining patterns of samples with positive results, patient antibodies react with indicator cells and the localization of patient antibodies is visualized by a second fluorescein antihuman IgG antibody evaluated under a fluorescence microscope. These patterns may provide additional information to rule out or further implicate a suspected condition and can guide the selection of additional testing for specific autoantibodies. 2

Figure 1. Use of ANA (IFA) and Specific Antibody Testing Cascade (Test Code 16814) Figure 1. Use of ANA (IFA) and Specific Antibody Testing Cascade (Test Code 16814) for the Diagnosis for the Diagnosis of Rheumatic Disease6-10 of Rheumatic Disease ANA Screen (IFA)

Negative

Positive

Autoimmune disease less likely. May consider rheumatoid arthritis disease testing.

Tier 1 Chromatin, dsDNA, RNP, Sm, and Sm/RNP antibodies

Titer and Pattern

Negative

Positive

Tier 2 Jo-1, Scl-70, SS-A, and SS-B antibodies Negative

Positive

Tier 3 Centromere B and ribosomal P antibodies Negative No evidence of rheumatic disease shown by analytes tested

Positive

Antibody Test Centromere B Ribosomal P

Antibody Test SS-A SS-B Scl-70 Jo-1 CREST Syndrome + –

Antibody Test dsDNA Chromatin Sm Sm/RNP RNP Sjögren’s Syndrome + + – –

Systemic Lupus Erythematosus + (high specificity) + (high sensitivity) + (high specificity)

Systemic Sclerosis – – + –

+ +

Mixed Connective Tissue Disease – – – + (high titer) + (high titer)

Polymyositis – – – +

Neuropsychiatric SLE – +

The acronym refers a syndrome defined bybypresence ofcalcinosis, calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, TheCREST acronym CRESTto refers to a syndrome defined presence of Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and and telangiectasia. dsDNA indicates DNA; Sm/RNP antibody, Smith/ribonucleoprotein SS-A and -B antibodies, telangiectasia. dsDNA indicates double-stranded double-stranded DNA; Sm/RNP antibody, Smith/ribonucleoprotein antibody; SS-A antibody; and -B antibodies, Sjögren's Sjögren’s Syndrome and B antibodies; antibody, scleroderma (topoisomerase I) antibody; Jo-1 antibody, histidyl-tRNA Syndrome AAand B antibodies; Scl-70 Scl-70 antibody, scleroderma (topoisomerase I) antibody; Jo-1 antibody, histidyl-tRNA synthetase antibody; and SLE,synthetase antibody; and SLE,lupus systemic lupus erythematosus. systemic erythematosus. was developed by Quest Diagnosticsbased based on 6-10.1-5. It is It provided for informational purposes only and is notonly intended This figure This wasfigure developed by Quest Diagnostics onreferences references is provided for informational purposes andasis not advice. A physician’s test selection interpretation, and patient management decisions should be b  ased on his/her education, intended asmedical medical advice. A physician’s testand selection and diagnosis, interpretation, diagnosis, and patient management decisions should be clinical expertise, and clinical assessment of the patient. based on his/her education, expertise, and assessment of the patient.

References 1. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med. 2000;124:71-81. 2. Satoh M, Chan EKL, Sobel ES, et al. Clinical implication of autoantibodies in patients with systemic rheumatic diseases.

Expert 1 Rev Clin Immunol. 2007;3:721-738. Figure above illustrates an example of an algorithm

(In the cell, Sm and RMP proteins form a complex.)

3. Stinton LM, Fritzler clinical approachtiers to autoantibody testing in systemic autoimmune approach with a MJ. set Aof reflexing for suspected If the first tierrheumatic yields adisorders. positive Autoimmun Rev. 2007;7:77-84.

result, the results are

rheumatic disease, more common group ofand autoimmune reported and the testing stops.clinics If the first tier criteria of antibody AM,aAlarcón GS, et al. Derivation validation of systemic lupus international collaborating classification 4. Petri M, Orbai for systemic lupus erythematosus. Arthritis Rheum. 2012;64:2677-2686. 6-10 diseases. With this algorithm, samples with a positive testing is negative, the testing reflexes to the 2nd tier, 5. Cappelli S, Randone SB. “To be or not to be,” ten years after: evidence for mixed connective tissue disease as a distinct entity.

IFA Semin resultArthritis are tested for 2012;41:589-598. five autoantibodies associated with Rheum.

which includes four autoantibodies associated with

systemic lupus erythematosus (SLE) and mixed connective

Sjögren’s Syndrome, systemic sclerosis, and polymyositis:

tissue disease: dsDNA, chromatin (nucleosomal), Smith

SS-A, SS-B, Scl-70, and Jo-I antibodies. If a positive result

(Sm), ribonuclear protein (RNP), and Sm/RNP antiodies.

is determined for any of these autoantibodies, the results 3

Table 1. Common Signs and Symptoms of Autoimmune Rheumatic and Related Diseasesa,b Sign or Symptom

Gout

JIA

MCTD

PM/DM

Pseudogout

RA

Sarcoidosis

Joint-/muscle-related Joint pain, stiffness, or inflammation Muscle weakness Myalgia Skin-/hair-related Alopecia Rash Raynaud’s phenomenon Skin lesions

c

General Anorexia Cough Ear involvement

d

Eye involvement Fatigue Fever GI involvement Malaise Nasal symptoms Nervous system involvement Respiratory involvement Weight loss Other Adenopathy Anemia Dysphagia Swelling of hands (Continued)

4

Table 1. Common Signs and Symptoms of Autoimmune Rheumatic and Related Diseasesa (Continued) Sign or Symptom

SjS

SLE

SpA AS

ReA

PsA

EA

SSc

Systemic Vasculitis GPA

EGPA

MPA

Joint-/muscle-related Joint pain, stiffness, or inflammation Muscle weakness Myalgia Skin-/hair-related Alopecia Rash Raynaud’s phenomenon Skin lesions General Anorexia Cough Ear involvement

d

Eye involvement Fatigue Fever GI involvement Malaise Nasal symptoms Nervous system involvement Respiratory involvement Weight loss Other Adenopathy Anemia Dysphagia Swelling of hands indicates common; indicates less common but not rare. AS=ankylosing spondylitis, EA=enteropathic (inflammatory bowel disease-associated) arthritis, EGPA=eosinophilic granulomatosis with polyangiitis, GPA=granulomatosis with polyangiitis, JIA=juvenile idiopathic arthritis, MCTD=mixed connective tissue disease, MPA=microscopic polyangiitis, PM/DM=polymyositis/dermatomyositis; RA=rheumatoid arthritis, PsA=psoriatic arthritis, ReA=reactive arthritis, SjS=Sjögren’s Syndrome, SLE=systemic lupus erythematosus, SpA=spondyloarthropathies, SSc=systemic sclerosis. b This is not a complete list of signs and symptoms; some conditions have more signs and symptoms than could be presented here. c In dermatomyositis. d External ear in gout; middle ear in GPA. a

5

are reported and the testing stops. If 2nd tier antibodies are negative, the testing continues with a reflex to 3rd tier. The 3rd tier includes two autoantibodies associated with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) and neuropsychiatric SLE: centromere B and ribosomal P antibodies.

Case Studies

1

SLE

A 32-year-old Caucasian woman presented for an initial visit because of soreness in her hands. This soreness began six weeks previously, with no history of injury or prior pain

tier will stop

and soreness. The middle fingers of both hands (fingers

the reflex testing to the next tier, it does not rule out the

3 and 4) were swollen and tender, and she had trouble

presence of additional clinically relevant autoantibodies,

making a fist and opening jars. Other joints were stiffer

including those in the next tier or other autoantibodies

than usual, especially her knees, ankles, and feet. She had

outside the algorithm. Additionally, negative results for all

difficulty getting out of bed in the morning, with joint

three tiers do not rule out an autoimmune disease, as not

stiffness and painful walking lasting many hours. The patient

all autoimmune diseases are represented in this algorithm.

denied fevers, chills, and any recent febrile illnesses, yet

Note that the clinician can request full reporting through

reported feeling especially tired since this issue began. She

all three tiers, especially if a rheumatic autoimmune disease

had been taking over-the-counter naproxen for “some”

is strongly suspected.

relief. She denied rash, dry eyes/mouth, sun sensitivity,

Although a positive result in the 1 or 2 st

nd

11

Clinical suspicion and correlation are generally necessary to proceed with additional testing for other specific antibodies beyond the algorithm of the most common rheumatic diseases. For example, a positive ANA IFA is a diagnostic criterion for drug-induced lupus, polymyositis/ dermatomyositis, other forms of idiopathic inflammatory myopathy, and oligoarticular juvenile chronic arthritis. A positive ANA result is also consistent with organ-specific autoimmune diseases, including thyroid diseases (eg, Hashimoto’s thyroiditis, Grave’s disease), gastrointestinal diseases (eg, autoimmune hepatitis, primary biliary

trouble

swallowing,

and

cardiovascular

symptoms.

The patient’s medical history was unremarkable. Her medication list included birth control pills and she denied any medication allergies. Her family history was significant for thyroid disease (mother and older sister) and type 1 diabetes (father); social history included smoking ½ pack per day for the past 18 years. She had no surgical history, and a review of systems was unremarkable (Table 2). Table 2. SLE Patient Vital Signs and Physical Examination Height

5'6"

Weight

129 lb

BMI

21

A positive ANA result alone is not diagnostic of an

HEENT

Normal

autoimmune disease. The prevalence of ANAs in healthy

Neck Adenopathy

None

Heart/Lung Auscultation

Normal

Abdomen

Benign

Hands

Boggy, tender, warm 3rd and 4th metacarpophalangeal joints on the right, minimally same on the left; rest of joints normal

Skin

No rash

cholangitis, inflammatory bowel disease), and pulmonary disease (eg, idiopathic pulmonary fibrosis).

individuals is about 3-15%.11 The production of these autoantibodies is strongly age-dependent, and increases to 10-37% in healthy persons over 65. Even healthy people with viral infections can have a positive ANA, albeit for a short time. Patients with infectious diseases may also test positive for ANA. These include viral infections (hepatitis C, parvovirus), bacterial infections (tuberculosis), and parasitic infections (schistosomiasis). Certain medications and some lymphomas may also cause a positive ANA.

The patient underwent laboratory testing using with the ANA IFA with 3rd tier specific autoantibody reflex cascade (Figure 1). The results were positive for ANA IFA at a titer of

6

1:160 with a mixed speckled and homogeneous pattern.

The results of routine laboratory testing (complete

The 1 tier of testing was positive for dsDNA, chromatin,

blood cell count, sedimentation rate, and comprehensive

and Sm antibodies, which pointed to a diagnosis of

metabolic panel) were normal. The ANA IFA was positive

SLE. The patient was then referred to a rheumatologist

at a titer of 1:320 with speckled pattern. First tier cascade

who confirmed the diagnosis of SLE, and started her on

testing (Figure 1) was negative, yet the 2nd tier was positive

hydroxychloroquine. The primary care physician continues

for SS-A and SS-B. Rheumatoid factor was ordered and

to work collaboratively with the rheumatologist to manage

was positive (≥14 IU/mL). The labs indicated a diagnosis of

this patient.

Sjögren’s Syndrome.

st

After the diagnosis of Sjögren’s Syndrome, the patient was referred to a rheumatologist for further evaluation

2

Sjögren’s Syndrome

A 58-year-old Japanese woman presented to the clinic complaining of a very dry mouth and thirst that had been going on for months and was now worsening. She was worried about a diagnosis of diabetes mellitus (T2DM) due to her family history (ie, parents had T2DM). The patient

and treatment. Consultation confirmed the diagnosis and she was started on prednisone 10 mg daily and hydroxychloroquine 200 mg daily. She was to follow-up with the rheumatologist for further definitive treatment. The primary care physician continues to work collaboratively with the rheumatologist to manage this patient.

had trouble speaking normally without constantly drinking water and found it difficult to chew and swallow food. Upon further questioning, she reported that she also had dry eyes but no polyuria or weight loss, no swollen salivary glands, no joint pain, and no skin changes (Table 3). Her medical history was significant for the deep vein thrombosis and for major depressive disorder, which had been treated and was now in remission. She had no medications or allergies to such. In addition to T2DM, her family history was positive for hypertension (parents), hyperlipidemia (parents), and rheumatoid arthritis (brother). The patient had never smoked cigarettes and the review of systems was unremarkable. On examination, the patient appeared comfortable and in no pain; vital signs were normal. Table 3. Sjögren’s Patient Vital Signs and Physical Examination

3

Autoimmune Hepatitis

A 43-year-old woman presented with a 5-month history of unintentional weight loss (13.2 lb), anorexia, irritability, malaise and generalized pruritus. On physical examination, she was mildly icteric with numerous scratch marks, palmar erythema, and hepatosplenomegaly; vital signs and physical exam were unremarkable (Table 4). Laboratory results showed a low hemoglobin level (8 g/dL) with a normal white-cell count and an elevated erythrocyte sedimentation rate (140 mm/h; normal ≤ 20 mm/h). The prothrombin time was prolonged, yet urea and electrolytes, calcium and phosphate concentrations were normal. Although the serum albumin was normal (4.1 g/dL), the following were elevated: total serum

Height

5'1"

proteins (9.3 g/dL; normal range 6.1-8.1 g/dL), serum

Weight

121 lb

bilirubin (1.8 mg/dL; normal range 0.2-1.2 mg/dL), alanine

BMI

23

transaminase (152 IU/L; normal range 7-55 IU/L), and

HEENT

Dry mouth and eyes; leathery tonge; no partoid swelling

aspartate transaminase (164 IU/L; normal range 8-48 IU/L).

Neck Adenopathy

None

Heart/Lung Auscultation

range 45-115 IU/L). Her serum immunoglobulins showed

Normal

Abdomen

Benign

Hands

Normal

Skin Normal

The alkaline phosphatase level was normal (83 IU/L; normal an increased IgG level (44 g/L; normal range 7.2-19.0 g/L) with normal IgA and IgM levels. Antinuclear antibodies by IFA were strongly positive (titer 1:320) in a homogeneous pattern, and 1st tier autoantibody 7

Table 4. Hepatitis Patient Vital Signs and Physical Examination

titer 1:320 was a definitive diagnosis. The patient was therefore started on prednisolone (30 mg/day) and vitamin

Height

5'7"

K, and showed dramatic improvement. Her serum bilirubin,

Weight

111 lb

transaminases, and prothrombin time returned to normal

BMI

17.4

over the next two weeks. A diagnostic liver biopsy showed

HEENT

Normal

chronic active hepatitis with cirrhosis. She was continued

Neck Adenopathy

None

on prednisolone (15 mg/day) and is fully reassessed every

Heart/Lung Auscultation

Normal

Abdomen

Distended; liver palpable below costal margin

Hands

Normal

Skin

Mildly icteric; pruritic with scratch marks on various locations including forearms, abdomen, and thighs; palmar erythema on both hands

six months, including repeat liver biopsy as appropriate.

Conclusion An ANA IFA cascade with reflex to specific testing has clinical significance in the proper setting. A positive

testing was revealed positive for only dsDNA antibodies.

ANA IFA by itself can show pre-clinical autoimmune

The ANA IFA also showed cytoplasmic staining of actin

disease, yet utilizing the ANA IFA cascade with reflex

elements. Testing was positive for antibodies to smooth

to specific testing can lead to early diagnosis and early

muscle consistent with autoimmune hepatitis. Hepatitis B

treatment of potentially devastating diseases, putting some

surface antigen and hepatitis C antibody was absent and

patients in remission. Test results should be interpreted in

alpha fetoprotein was not detected. The immunological

a clinical context that includes a history and physical, basic

picture and absence of hepatitis B and C infection strongly

chemistry panel, imaging studies, and assessment of signs

favored a diagnosis of autoimmune hepatitis. The ANA

and symptoms.

References 1. Hajj-ali RA. Introduction to rheumatic diseases. https://www.merckmanuals.com/professional/musculoskeletal-and-connective-tissue-disorders/autoimmune-rheumatic disorders/introduction-to-autoimmune-rheumatic-disorders. Accessed August 29, 2016. 2. Autoimmune disease in women. American Autoimmune Related Diseases Association, Inc. http://www.aarda.org/autoimmune-information/autoimmune-disease-in-women/. Accessed August 29, 2016. 3. Suarez-Almazor M, Gonzalez-Lopez L, Gamez-Nava I, et al. Utilization and predictive value of laboratory tests in patients referred to rheumatologists by primary care physicians. J Rheumatol. 1998; 25:1980-1985. 4. American College of Rheumatology Position Statement: Methodology of testing for antinuclear antibodies. Published January 2009. Updated August 2015. http://www.rheumatology.org/Portals/0/Files/Methodology%20of%20Testing%20Antinuclear%20Antibodies%20Position%20Statement.pdf. Accessed August 29, 2016. 5. Salzman BE, Nevin JE, Newman JH. A primary care approach to the use and interpretation of common rheumatologic tests. Clinics in Family Practice. 2005; 7(2):335-358. 6. Kavanaugh A, Tomar R, Reveille J, et al. Guidelines for the clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med. 2000; 124:71-81. 7. Satoh M, Chan EKL, Sobel ES, et al. Clinical implication of autoantibodies in patients with systemic rheumatic diseases. Expert Rev Clin Immunol. 2007; 3:721-738. 8. Stinton LM, Fritzler MJ. A clinical approach to autoantibody testing in systemic autoimmune rheumatic disorders. Autoimmun Rev. 2007; 7:77-84. 9. Petri M, Orbai AM, Alarcón GS, et al. Derivation and validation of systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012; 64:2677-2686. 10. Cappelli S, Randone SB. “To be or not to be,” ten years after: Evidence for mixed connective tissue disease as a distinct entity. Semin Arthritis Rheum. 2012; 41:589-98. 1. Gutierrez-Adrianzen OA, Koutouzov S, Mota RM, et al. Diagnostic value of anti-nucleosome antibodies in the assessment of disease activity of systemic lupus 1 erythematosus: a prospective study comparing anti-nucleosome with anti-dsDNA antibodies. J Rheumatol. 2006; 33:1538-1544. MI6203 12/2016